Abstract
Objectives
To identify an efficient in vitro refolding method to generate highly active His6-tagged scorpion toxin antitumor-analgesic peptide (AGAP) isolated from Escherichia coli inclusion bodies.
Results
N- and C-Terminal His6-tagged recombinant (r) AGAP (NHis6-rAGAP and CHis6-rAGAP, respectively) were expressed in E. coli; the purification and refolding conditions were optimized. CHis6-rAGAP, but not NHis6-rAGAP, exhibited significant in vitro antihepatoma activity that was much greater than that of rAGAP produced using SUMO fusion technology (IC50, 0.4 ± 0.08 vs. 1.8 ± 0.3 μM). CHis6-rAGAP also showed significant inhibition of tumor growth in a mouse xenograft model of human hepatoma and inhibition of neuronal excitability, demonstrated by blockage of voltage-sensitive tetrodotoxin-resistant (TTX-R) sodium currents in acute isolated dorsal root ganglion neurons.
Conclusions
This refolding protocol optimized for C-terminal His6-tagged scorpion rAGAP is potentially applicable to similar long-chain and cysteine-rich toxins.
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Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (Nos. 81302694, 81473377, 81573837), the Natural Science Foundation of Jiangsu Province (BK2012492, BK20131038, BK20140049), Shanghai Municipal Natural Science Foundation (No. 13ZR1448900) and the Natural Science Foundation of Shanghai (No. 12ZR1415600).
Supporting information
Supplementary Methods: Optimization of rAGAP refolding and purification; Preparation of human hepatoma xenografts in mice and assay of antitumor activity of rAGAP in vivo.
Supplementary Table 1—Purification and recovery of CHis6-rAGAPa.
Supplementary Fig. 1—Construction of the plasmid pET29a/CHis6-rAGAP.
Supplementary Fig. 2—SDS-PAGE and HPLC analyses of expression, purification, and refolding of CHis6-rAGAP a SDS-PAGE analysis of CHis6-rAGAP expression and inclusion body preparation.
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Qingxin Cao and Wuguang Lu have contributed equally to this work.
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Cao, Q., Lu, W., Cai, X. et al. In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli . Biotechnol Lett 37, 2461–2466 (2015). https://doi.org/10.1007/s10529-015-1936-8
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DOI: https://doi.org/10.1007/s10529-015-1936-8